Evaluating the effects of long term exposure to environmental relevant concentrations of real life mixtures of persistent organic pollutants (POPs) in the zebrafish model.

Persistent organic pollutants (POPs) have been widely distributed throughout the world for decades and while the use of some have been phased out, others are still being utilised and their levels are rising in biota. Levels detected in tissue and blood samples have caused for a growing concern for their potential effects on humans and wildlife and many effects studies have been performed. Focus for research has, however, mainly been on high concentrations of single compounds, although it is the realistic levels of real life mixtures of compounds found in the environment that may possess such a threat. In this study, we have investigated the long term effects of environmentally relevant levels of real life mixtures of POPs using the zebrafish as a biomonitor organism. Burbots (Lota lota) from two sites within the same freshwater system in Norway, Lake Mjøsa and Lake Losna, with different history of pollution, were captured and POPs extracted from the liver oil. Zebrafish were exposed indirectly, by exposing their live-food, from start of feeding and until sexual maturation to either the Losna mixture or to one of three dose levels of the Mjøsa mixture. Survival was monitored throughout the experiment while other demographic variables such as growth and sex ratio were evaluated at the time of sexual maturity. Traditional (protein) biomarkers for EROD activity and vitellogenin induction were also measured in addition to organ-specific differences in gene expression pattern using microarray analysis. Exposure resulted in significant lower survival rate and increased growth of the fish, while a small but significant induction was observed in the EROD activity. Analysis of gene expression patterns revealed small changes in mRNA levels though differences were clearly seen. The microarray assay indicated oestrogenic effects of both of the mixtures, in addition to other endocrine disrupting effects related to the steroid- and thyroid hormones. We conclude that long term exposure to real life mixtures of POPs caused direct morphological and phenotypic changes and effects systems related to development and reproduction even at low levels found in the environment, and thus may pose a potential health risk for humans and wildlife living in exposed areas or in other ways being frequently exposed to such mixtures of toxins

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro

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iPSC-derived liver organoids and inherited bleeding disorders : potential and future perspectives

Funding: This work was made possible with funding from the Norwegian Research Council (325869) and the South-Eastern Norway Regional Health Authority (2019071).The bleeding phenotype of hereditary coagulation disorders is caused by the low or undetectable activity of the proteins involved in hemostasis, due to a broad spectrum of genetic alterations. Most of the affected coagulation factors are produced in the liver. Therefore, two-dimensional (2D) cultures of primary human hepatocytes and recombinant overexpression of the factors in non-human cell lines have been primarily used to mimic disease pathogenesis and as a model for innovative therapeutic strategies. However, neither human nor animal cells fully represent the hepatocellular biology and do not harbor the exact genetic background of the patient. As a result, the inability of the current in vitro models in recapitulating the in vivo situation has limited the studies of these inherited coagulation disorders. Induced Pluripotent Stem Cell (iPSC) technology offers a possible solution to overcome these limitations by reprogramming patient somatic cells into an embryonic-like pluripotent state, thus giving the possibility of generating an unlimited number of liver cells needed for modeling or therapeutic purposes. By combining this potential and the recent advances in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, it allows for the generation of autologous and gene corrected liver cells in the form of three-dimensional (3D) liver organoids. The organoids recapitulate cellular composition and organization of the liver, providing a more physiological model to study the biology of coagulation proteins and modeling hereditary coagulation disorders. This advanced methodology can pave the way for the development of cell-based therapeutic approaches to treat inherited coagulation disorders. In this review we will explore the use of liver organoids as a state-of-the-art methodology for modeling coagulation factors disorders and the possibilities of using organoid technology to treat the disease.Peer reviewe

LIGHT/TNFSF14 is increased in patients with type 2 diabetes mellitus and promotes islet cell dysfunction and endothelial cell inflammation in vitro

Published version. Source at http://dx.doi.org/10.1007/s00125-016-4036-y Aims/hypothesis: Activation of inflammatory pathways is involved in the pathogenesis of type 2 diabetes mellitus. On the basis of its role in vascular inflammation and in metabolic disorders, we hypothesised that the TNF superfamily (TNFSF) member 14 (LIGHT/TNFSF14) could be involved in the pathogenesis of type 2 diabetes mellitus. Methods: Plasma levels of LIGHT were measured in two cohorts of type 2 diabetes mellitus patients (191 Italian and 40 Norwegian). Human pancreatic islet cells and arterial endothelial cells were used to explore regulation and relevant effects of LIGHT in vitro. Results: Our major findings were: (1) in both diabetic cohorts, plasma levels of LIGHT were significantly raised compared with sex- and age-matched healthy controls (n = 32); (2) enhanced release from activated platelets seems to be an important contributor to the raised LIGHT levels in type 2 diabetes mellitus; (3) in human pancreatic islet cells, inflammatory cytokines increased the release of LIGHT and upregulated mRNA and protein levels of the LIGHT receptors lymphotoxin β receptor (LTβR) and TNF receptor superfamily member 14 (HVEM/TNFRSF14); (4) in these cells, LIGHT attenuated the insulin release in response to high glucose at least partly via pro-apoptotic effects; and (5) in human arterial endothelial cells, glucose boosted inflammatory response to LIGHT, accompanied by an upregulation of mRNA levels of HVEM (also known as TNFRSF14) and LTβR (also known as LTBR). Conclusions/interpretation: Our findings show that patients with type 2 diabetes mellitus are characterised by increased plasma LIGHT levels. Our in vitro findings suggest that LIGHT may contribute to the progression of type 2 diabetes mellitus by attenuating insulin secretion in pancreatic islet cells and by contributing to vascular inflammation

SARS-CoV-2 vaccines are not associated with hypercoagulability in apparently healthy people

Background: SARS-CoV-2 adenoviral vector DNA vaccines have been linked to the rare but serious thrombotic postvaccine complication vaccine-induced immune thrombotic thrombocytopenia. This has raised concerns regarding the possibility of increased thrombotic risk after any SARS-CoV-2 vaccines. Objectives: To investigate whether SARS-CoV-2 vaccines cause coagulation activation leading to a hypercoagulable state. Methods: This observational study included 567 health care personnel; 521 were recruited after the first dose of adenoviral vector ChAdOx1-S (Vaxzevria, AstraZeneca) vaccine and 46 were recruited prospectively before vaccination with a messenger RNA (mRNA) vaccine, either Spikevax (Moderna, n = 38) or Comirnaty (Pfizer-BioNTech, n = 8). In the mRNA group, samples were acquired before and 1 to 2 weeks after vaccination. In addition to the prevaccination samples, 56 unvaccinated blood donors were recruited as controls (total n = 102). Thrombin generation, D-dimer levels, and free tissue factor pathway inhibitor (TFPI) levels were analyzed. Results: No participant experienced thrombosis, vaccine-induced immune thrombotic thrombocytopenia, or thrombocytopenia (platelet count 9 /L) 1 week to 1 month postvaccination. There was no increase in thrombin generation, D-dimer level, or TFPI level in the ChAdOx1-S vaccine group compared with controls or after the mRNA vaccines compared with baseline values. Eleven of 513 (2.1%) participants vaccinated with ChAdOx1-S had anti-PF4/polyanion antibodies without a concomitant increase in thrombin generation. Conclusion: In this study, SARS-CoV-2 vaccines were not associated with thrombosis, thrombocytopenia, increased thrombin generation, D-dimer levels, or TFPI levels compared with baseline or unvaccinated controls. These findings argue against the subclinical activation of coagulation post-COVID-19 vaccination

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility

<p>Abstract</p> <p>Background</p> <p>Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231).</p> <p>Methods</p> <p>Stable cell lines with TFPI (both α and β) and only TFPIβ downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion.</p> <p>Results</p> <p>Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIβ was downregulated, revealing a novel function of this isoform in cancer metastasis.</p> <p>Conclusions</p> <p>Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.</p

TFPIα and TFPIβ are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity

<p>Abstract</p> <p>Background</p> <p>Tissue factor (TF) pathway inhibitor-1 (TFPI) is expressed in several malignant tissues- and cell lines and we recently reported that it possesses anti-tumor effects in breast cancer cells, indicating a biological role of TFPI in cancer. The two main splice variants of TFPI; TFPIα and TFPIβ, are both able to inhibit TF-factor VIIa (FVIIa) activity in normal cells, but only TFPIα circulates in plasma. The functional importance of TFPIβ is therefore largely unknown, especially in cancer cells. We aimed to characterize the expression and function of TFPIα, TFPIβ, and TF in a panel of tumor derived breast cancer cell lines in comparison to normal endothelial cells.</p> <p>Methods</p> <p>TFPIα, TFPIβ, and TF mRNA and protein measurements were conducted using qRT-PCR and ELISA, respectively. Cell-associated TFPI was detected after phosphatidylinositol-phospholipase C (PI-PLC) and heparin treatment by flow cytometry, immunofluorescence, and Western blotting. The potential anticoagulant activity of cell surface TFPI was determined in a factor Xa activity assay.</p> <p>Results</p> <p>The expression of both isoforms of TFPI varied considerably among the breast cancer cell lines tested, from no expression in Sum149 cells to levels above or in the same range as normal endothelial cells in Sum102 and MDA-MB-231 cells. PI-PLC treatment released both TFPIα and TFPIβ from the breast cancer cell membrane and increased TF activity on the cell surface, showing TF-FVIIa inhibitory activity of the glycosylphosphatidylinositol- (GPI-) anchored TFPI. Heparin treatment released TFPIα without decreasing the cell surface levels, thus indicating the presence of intracellular storage pools of TFPIα in the breast cancer cells.</p> <p>Conclusion</p> <p>GPI-attached TFPI located at the surface of breast cancer cells inhibited TF activity and could possibly reduce TF signaling and breast cancer cell growth locally, indicating a therapeutic potential of the TFPIβ isoform.</p

Indirect regulation of TFPI-2 expression by miR-494 in breast cancer cells

Abstract TFPI-2 has been shown to be involved in breast cancer pathogenesis by inhibiting extracellular matrix degradation, and low levels are associated with disease progression. As microRNA-494 (miR-494) protects against breast cancer progression, we investigated whether miR-494 is involved in the regulation of TFPI-2 in MCF-7 breast cancer cells. TFPI-2 mRNA and protein levels increased after transfection with miR-494 mimic, and TFPI-2 mRNA and miR-494 levels correlated positively in tumors from breast cancer patients. No specific binding sites for miR-494 in the 3′-untranslated region (UTR) of TFPI2 were identified; however, miR-494 was predicted in silico to bind 3′-UTR of the transcription factors AHR and ELF-1, which have potential binding sites in the TFPI2 promoter. ELF-1 mRNA was downregulated whereas AHR mRNA levels were upregulated after transfection with miR-494 mimic. Knockdown of ELF-1 and AHR increased and reduced TFPI-2 mRNA levels, respectively. Increased luciferase activity was seen when TFPI-2 promoter constructs containing the potential AHR or ELF-1 binding sites were co-transfected with miR-494 mimic. In conclusion, TFPI-2 mRNA levels were upregulated by miR-494 in MCF-7 breast cancer cells most likely by an indirect association where miR-494 targeted the transcription factors AHR and ELF-1. This association was supported in a breast cancer cohort

Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform

OBJECTIVES: Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD.METHODS: We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days.RESULTS: Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results.CONCLUSIONS: Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.</p